Soluble protein antigens are internalized by antigen presenting cells and processed (denatured and/or partially degraded) into peptides, which can associate with Class II molecules and activate T cells. A similar mechanism has been recently proposed for endogenously expressed, Class I restricted antigens, suggesting that antigen processing may be a general feature of MHC-restricted antigen presentation. However, a bias has been observed such that Class I restricted antigens require endogenous expression, whereas Class II molecules can associate with exogenous, internalized antigens. This difference suggests that there may be distinct pathways for antigen processing and association with Class I vs. Class II molecules. One explanation would be if Class II molecules were sequestered with recently processed exogenous antigens in an internal cellular compartment in the absence of Class I molecules. This suggestion would imply that only recently synthesized or recycled Ia molecules can associate with newly processed antigen and it raises the possibility that the interaction of Ia with invariant chain (Ii) during biosynthesis may be important or these processes. Two independent approaches will be used to test these predictions. First, Ia will be transfected with and without Ii into cells which do not express either of these molecules. In addition, Ii-negative variants will be selected from Ia, Ii-positive cells by transfection of an anti-sense RNA encoding Ii construct. These pairs of cells expressing Ia + Ii will be assayed for their ability to process and present antigen to Class II restricted T cells. Second, Ia will be placed under control of the inducible E. coli lac operator and repressor to examine the relative contribution of newly synthesized Ia compared to cell surface Ia in the association and presentation of intact antigens. Together, these experiments should determine the importance of intracellular Ia in antigen presentation.